The Specificity of Controlled Protein Disorder in the Photoprotection of?Plants

Light-harvesting pigment-protein complexes of photosystem II of plants have a dual function: they efficiently use absorbed energy for photosynthesis at limiting sunlight intensity and dissipate the excess energy at saturating intensity for photoprotection. Recent single-molecule spectroscopy studies on the trimeric LHCII complex showed that environmental control of the intrinsic protein disorder could in principle explain the switch between their light-harvesting and photoprotective conformations in vivo. However, the validity of this proposal depends strongly on the specificity of the protein dynamics. Here, a similar study has been performed on the minor monomeric antenna complexes of photosystem II (CP29, CP26, and CP24). Despite their high structural homology, similar pigment content and organization compared to LHCII trimers, the environmental response of these proteins was found to be rather distinct. A much larger proportion of the minor antenna complexes were present in permanently weakly fluorescent states under most conditions used; however, unlike LHCII trimers the distribution of the single-molecule population between the strongly and weakly fluorescent states showed no significant sensitivity to low pH, zeaxanthin, or low detergent conditions. The results support a unique role for LHCII trimers in the regulation of light harvesting by controlled fluorescence blinking and suggest that any contribution of the minor antenna complexes to photoprotection would probably involve a distinct mechanism.     

Substrate Specificity of Purified Recombinant Human β-Carotene 15,15′-Oxygenase (BCO1)

Humans cannot synthesize vitamin A and thus must obtain it from their diet. β-Carotene 15,15′-oxygenase (BCO1) catalyzes the oxidative cleavage of provitamin A carotenoids at the central 15–15′ double bond to yield retinal (vitamin A). In this work, we quantitatively describe the substrate specificity of purified recombinant human BCO1 in terms of catalytic efficiency values (k_cat/K_m). The full-length open reading frame of human BCO1 was cloned into the pET-28b expression vector with a C-terminal polyhistidine tag, and the protein was expressed in the Escherichia coli strain BL21-Gold(DE3). The enzyme was purified using cobalt ion affinity chromatography. The purified enzyme preparation catalyzed the oxidative cleavage of β-carotene with a V_max = 197.2 nmol retinal/mg BCO1 × h, K_m = 17.2 μm and catalytic efficiency k_cat/K_m = 6098 m⁻¹ min⁻¹. The enzyme also catalyzed the oxidative cleavage of α-carotene, β-cryptoxanthin, and β-apo-8′-carotenal to yield retinal. The catalytic efficiency values of these substrates are lower than that of β-carotene. Surprisingly, BCO1 catalyzed the oxidative cleavage of lycopene to yield acycloretinal with a catalytic efficiency similar to that of β-carotene. The shorter β-apocarotenals (β-apo-10′-carotenal, β-apo-12′-carotenal, β-apo-14′-carotenal) do not show Michaelis-Menten behavior under the conditions tested. We did not detect any activity with lutein, zeaxanthin, and 9-cis-β-carotene. Our results show that BCO1 favors full-length provitamin A carotenoids as substrates, with the notable exception of lycopene. Lycopene has previously been reported to be unreactive with BCO1, and our findings warrant a fresh look at acycloretinal and its alcohol and acid forms as metabolites of lycopene in future studies.     

The Cytoplasmic Domain of the SARS-CoV-2 Envelope Protein Assembles into a β-Sheet Bundle in Lipid Bilayers

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope (E) protein forms a pentameric ion channel in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment (ERGIC) of the infected cell. The cytoplasmic domain of E interacts with host proteins to cause virus pathogenicity and may also mediate virus assembly and budding. To understand the structural basis of these functions, here we investigate the conformation and dynamics of an E protein construct (residues 8–65) that encompasses the transmembrane domain and the majority of the cytoplasmic domain using solid-state NMR. ¹³C and ¹⁵N chemical shifts indicate that the cytoplasmic domain adopts a β-sheet-rich conformation that contains three β-strands separated by turns. The five subunits associate into an umbrella-shaped bundle that is attached to the transmembrane helices by a disordered loop. Water-edited NMR spectra indicate that the third β-strand at the C terminus of the protein is well hydrated, indicating that it is at the surface of the β-bundle. The structure of the cytoplasmic domain cannot be uniquely determined from the inter-residue correlations obtained here due to ambiguities in distinguishing intermolecular and intramolecular contacts for a compact pentameric assembly of this small domain. Instead, we present four structural topologies that are consistent with the measured inter-residue contacts. These data indicate that the cytoplasmic domain of the SARS-CoV-2 E protein has a strong propensity to adopt β-sheet conformations when the protein is present at high concentrations in lipid bilayers. The equilibrium between the β-strand conformation and the previously reported α-helical conformation may underlie the multiple functions of E in the host cell and in the virion.     

“Substrate Inhibition” Is One of the Aspects of Substrate Specificity in Vertebrate and Invertebrate Cholinesterases

An analytical review is performed of the literature data on the hydrolysis rate, affinity of substrate to active center, and constants of the substrate inhibition (K _ss) at hydrolysis of the choline (acetyl-, propyonyl-, butyrylcholine, acetyl--methylcholine) and/or of corresponding thiocholine substrates by 59 cholinesterases from 49 different animals (chordate, insects, molluscs, nematodes). The characteristic peculiarities of enzymes from different groups of animals are revealed. The absence of regular changes of parameters of the cholinesterase substrate specificity in the course of evolutionary development is shown.     

Pancreatic β-Cell Death due to Pdx-1 Deficiency Requires Multi-BH Domain Protein Bax but Not Bak

Diabetes develops in Pdx1-haploinsufficient mice due to an increase in β-cell death leading to reduced β-cell mass and decreased insulin secretion. Knockdown of Pdx1 gene expression in mouse MIN6 insulinoma cells induced apoptotic cell death with an increase in Bax activation and knockdown of Bax reduced apoptotic β-cell death. In Pdx1 haploinsufficient mice, Bax ablation in β-cells increased β-cell mass, decreased the number of TUNEL positive cells and improved glucose tolerance after glucose challenge. These changes were not observed with Bak ablation in Pdx1-haploinsufficient mice. These results suggest that Bax mediates β-cell apoptosis in Pdx1-deficient diabetes.     

The Thalidomide-Binding Domain of Cereblon Defines the CULT Domain Family and Is a New Member of the β-Tent Fold

Despite having caused one of the greatest medical catastrophies of the last century through its teratogenic side-effects, thalidomide continues to be an important agent in the treatment of leprosy and cancer. The protein cereblon, which forms an E3 ubiquitin ligase compex together with damaged DNA-binding protein 1 (DDB1) and cullin 4A, has been recently indentified as a primary target of thalidomide and its C-terminal part as responsible for binding thalidomide within a domain carrying several invariant cysteine and tryptophan residues. This domain, which we name CULT (cereblon domain of unknown activity, binding cellular ligands and thalidomide), is also found in a family of secreted proteins from animals and in a family of bacterial proteins occurring primarily in δ-proteobacteria. Its nearest relatives are yippee, a highly conserved eukaryotic protein of unknown function, and Mis18, a protein involved in the priming of centromeres for recruitment of CENP-A. Searches for distant homologs point to an evolutionary relationship of CULT, yippee, and Mis18 to proteins sharing a common fold, which consists of two four-stranded β-meanders packing at a roughly right angle and coordinating a zinc ion at their apex. A β-hairpin inserted into the first β-meander extends across the bottom of the structure towards the C-terminal edge of the second β-meander, with which it forms a cradle-shaped binding site that is topologically conserved in all members of this fold. We name this the β-tent fold for the striking arrangement of its constituent β-sheets. The fold has internal pseudosymmetry, raising the possibility that it arose by duplication of a subdomain-sized fragment.     

Comprehensive Assessment of the Relationship Between Site−2 Specificity and Helix α2 in the Erbin PDZ Domain

PDZ domains are key players in signalling pathways. These modular domains generally recognize short linear C-terminal stretches of sequences in proteins that organize the formation of complex multi-component assemblies. The development of new methodologies for the characterization of the molecular principles governing these interactions is critical to fully understand the functional diversity of the family and to elucidate biological functions for family members. Here, we applied an in vitro evolution strategy to explore comprehensively the capacity of PDZ domains for specific recognition of different amino acids at a key position in C-terminal peptide ligands. We constructed a phage-displayed library of the Erbin PDZ domain by randomizing the binding site⁻² and adjacent residues, which are all contained in helix α2, and we selected for variants binding to a panel of peptides representing all possible position⁻² residues. This approach generated insights into the basis for the common natural class I and II specificities, demonstrated an alternative basis for a rare natural class III specificity for Asp⁻², and revealed a novel specificity for Arg⁻² that has not been reported in natural PDZ domains. A structure of a PDZ-peptide complex explained the minimum requirement for switching specificity from class I ligands containing Thr/Ser⁻² to class II ligands containing hydrophobic residues at position⁻². A second structure explained the molecular basis for the specificity for ligands containing Arg⁻². Overall, the evolved PDZ variants greatly expand our understanding of site⁻² specificities and the variants themselves may prove useful as building blocks for synthetic biology.     

The N-terminal Domain Tethers the Voltage-gated Calcium Channel β2e-subunit to the Plasma Membrane via Electrostatic and Hydrophobic Interactions

The β-subunit associates with the α₁ pore-forming subunit of high voltage-activated calcium channels and modulates several aspects of ion conduction. Four β-subunits are encoded by four different genes with multiple splice variants. Only two members of this family, β_2a and β_2e, associate with the plasma membrane in the absence of the α₁-subunit. Palmitoylation on a di-cysteine motif located at the N terminus of β_2a promotes membrane targeting and correlates with the unique ability of this protein to slow down inactivation. In contrast, the mechanism by which β_2e anchors to the plasma membrane remains elusive. Here, we identified an N-terminal segment in β_2e encompassing a cluster of positively charged residues, which is strictly required for membrane anchoring, and when transferred to the cytoplasmic β_1b isoform it confers membrane localization to the latter. In the presence of negatively charged phospholipid vesicles, this segment binds to acidic liposomes dependently on the ionic strength, and the intrinsic fluorescence emission maxima of its single tryptophan blue shifts considerably. Simultaneous substitution of more than two basic residues impairs membrane targeting. Coexpression of the fast inactivating R-type calcium channels with wild-type β_2e, but not with a β_2e membrane association-deficient mutant, slows down inactivation. We propose that a predicted α-helix within this domain orienting parallel to the membrane tethers the β_2e-subunit to the lipid bilayer via electrostatic interactions. Penetration of the tryptophan side chain into the lipidic core stabilizes the membrane-bound conformation. This constitutes a new mechanism for membrane anchoring among the β-subunit family that also sustains slowed inactivation.     

The C-terminal Domain of the Long Form of Cellular FLICE-inhibitory Protein (c-FLIPL) Inhibits the Interaction of the Caspase 8 Prodomain with the Receptor-interacting Protein 1 (RIP1) Death Domain and Regulates Caspase 8-dependent Nuclear Factor κB (NF-κB) Activation

Caspase 8 plays an essential role in the regulation of apoptotic and non-apoptotic signaling pathways. The long form of cellular FLICE-inhibitory protein (c-FLIP_L) has been shown previously to regulate caspase 8-dependent nuclear factor κB (NF-κB) activation by receptor-interacting protein 1 (RIP1) and TNF receptor-associated factor 2 (TRAF2). In this study, the molecular mechanism by which c-FLIP_L regulates caspase 8-dependent NF-κB activation was further explored in the human embryonic kidney cell line HEK 293 and variant cells barely expressing caspase 8. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone greatly diminished caspase 8-dependent NF-κB activation induced by Fas ligand (FasL) when c-FLIP_L, but not its N-terminal fragment c-FLIP(p43), was expressed. The prodomain of caspase 8 was found to interact with the RIP1 death domain and to be sufficient to mediate NF-κB activation induced by FasL or c-FLIP(p43). The interaction of the RIP1 death domain with caspase 8 was inhibited by c-FLIP_L but not c-FLIP(p43). Thus, these results reveal that the C-terminal domain of c-FLIP_L specifically inhibits the interaction of the caspase 8 prodomain with the RIP1 death domain and, thereby, regulates caspase 8-dependent NF-κB activation.     

Substrate inhibition and affinities of the glyoxysomal β-oxidation of sunflower cotyledons

During the glyoxysomal β-oxidation of long-chain acyl-CoAs, short-chain intermediates accumulate transiently (Kleiter and Gerhardt 1998, Planta 206: 125–130). The studies reported here address the underlying factors. The studies concentrated upon the aspects of (i) chain length specificity and (ii) metabolic regulation of the glyoxysomal β-oxidation of sunflower (Helianthus annuus L.) cotyledons. (i) Concentration-rate curves of the β-oxidation of acyl-CoAs of various chain lengths showed that the β-oxidation activity towards long-chain acyl-CoAs was higher than that towards short-chain acyl-CoAs at substrate concentrations <20 μM. At substrate concentrations >20 μM, long-chain acyl-CoAs were β-oxidized more slowly than short-chain acyl-CoAs because the β-oxidation of long-chain acyl-CoAs is subject to substrate inhibition which had already started at 5–10 μM substrate concentration and results from an inhibition of the multifunctional protein (MFP) of the β-oxidation reaction sequence. However, low concentrations of free long-chain acyl-CoAs are rather likely to exist within the glyoxysomes due to the acyl-CoA-binding capacity of proteins. Consequently, the β-oxidation rate towards a parent long-chain acyl-CoA will prevail over that towards the short-chain intermediates. (ii) Low concentrations (≤5 μM) of a long-chain acyl-CoA exerted an inhibitory effect on the β-oxidation rate of butyryl-CoA. Reversibility of the inhibition was observed as well as metabolization of the inhibiting long-chain acyl-CoA. Regarding the activities of the individual β-oxidation enzymes towards their C₄ substrates in the presence of a long-chain acyl-CoA, the MFP activity exhibited strong inhibition. This inhibition appears not to be due to the detergent-like physical properties of long-chain acyl-CoAs. The results of the studies, which are consistent with the observation that short-chain intermediates accumulate transiently during complete degradation of a long-chain acyl-CoA, suggest that the substrate concentration-dependent chain-length specificity of the β-oxidation and a metabolic regulation at the level of MFP are factors determining this transient accumulation. Received: 2 February 1999 / Accepted: 14 April 1999     

The Nonreceptor Protein Tyrosine Phosphatase PTP1B Binds to the Cytoplasmic Domain of N-Cadherin and Regulates the Cadherin–Actin Linkage

Cadherin-mediated adhesion depends on the association of its cytoplasmic domain with the actin-containing cytoskeleton. This interaction is mediated by a group of cytoplasmic proteins: α-and β- or γ- catenin. Phosphorylation of β-catenin on tyrosine residues plays a role in controlling this association and, therefore, cadherin function. Previous work from our laboratory suggested that a nonreceptor protein tyrosine phosphatase, bound to the cytoplasmic domain of N-cadherin, is responsible for removing tyrosine-bound phosphate residues from β-catenin, thus maintaining the cadherin–actin connection (Balsamo et al., 1996). Here we report the molecular cloning of the cadherin-associated tyrosine phosphatase and identify it as PTP1B. To definitively establish a causal relationship between the function of cadherin-bound PTP1B and cadherin-mediated adhesion, we tested the effect of expressing a catalytically inactive form of PTP1B in L cells constitutively expressing N-cadherin. We find that expression of the catalytically inactive PTP1B results in reduced cadherin-mediated adhesion. Furthermore, cadherin is uncoupled from its association with actin, and β-catenin shows increased phosphorylation on tyrosine residues when compared with parental cells or cells transfected with the wild-type PTP1B. Both the transfected wild-type and the mutant PTP1B are found associated with N-cadherin, and recombinant mutant PTP1B binds to N-cadherin in vitro, indicating that the catalytically inactive form acts as a dominant negative, displacing endogenous PTP1B, and rendering cadherin nonfunctional. Our results demonstrate a role for PTP1B in regulating cadherin-mediated cell adhesion.     

The Role of the Amino-Terminal β-Barrel Domain of the α and β Subunits in the Yeast F1-ATPase

The crystal structure of mitochondrial F₁-ATPase indicatesthat the and subunits fold into a structure defined by threedomains: the top -barrel domain, the middle nucleotide-binding domain,and the C-terminal -helix bundle domain (Abraham et al.1994); Bianchet et al., 1998). The -barrel domains of the and subunits form a crown structure at the top ofF₁, which was suggested to stabilize it (Abraham et al.1994). In this study. the role of the -barrel domain in the and subunits of the yeast Saccharomyces cerevisiae F₁,with regard to its folding and assembly, was investigated. The -barreldomains of yeast F₁ and subunits were expressedindividually and together in Escherichia coli. When expressedseperately, the -barrel domain of the subunit formed a largeaggregate structure, while the domain of the subunit waspredominately a monomer or dimer. However, coexpression of the -barreldomain of subunit domain. Furthermore, the two domains copurified incomplexes with the major portion of the complex found in a small molecularweight form. These results indicate that the -barrel domain of the and subunits interact specifically with each other and thatthese interactions prevent the aggregation of the -barrel domain of the subunit. These results mimic in vivo results and suggest thatthe interactions of the -barrel domains may be critical during thefolding and assembly of F₁.     

Revisiting the Nucleotide and Aminoglycoside Substrate Specificity of the Bifunctional Aminoglycoside Acetyltransferase(6′)-Ie/Aminoglycoside Phosphotransferase(2″)-Ia Enzyme

The bifunctional aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6′)-Ie/aminoglycoside phosphotransferase(2″)-Ia, or AAC(6′)-Ie/APH(2″)-Ia, is the major source of aminoglycoside resistance in Gram-positive bacterial pathogens. In previous studies, using ATP as the cosubstrate, it was reported that the APH(2″)-Ia domain of this enzyme is unique among aminoglycoside phosphotransferases, having the ability to inactivate an unusually broad spectrum of aminoglycosides, including 4,6- and 4,5-disubstituted and atypical. We recently demonstrated that GTP, and not ATP, is the preferred cosubstrate of this enzyme. We now show, using competition assays between ATP and GTP, that GTP is the exclusive phosphate donor at intracellular nucleotide levels. In light of these findings, we reevaluated the substrate profile of the phosphotransferase domain of this clinically important enzyme. Steady-state kinetic characterization using the phosphate donor GTP demonstrates that AAC(6′)-Ie/APH(2″)-Ia phosphorylates 4,6-disubstituted aminoglycosides with high efficiency (k_cat/K_m = 10⁵-10⁷ m⁻¹ s⁻¹). Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. We now show that phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and thus these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme. Taken together, our data show that the bifunctional enzyme efficiently phosphorylates only 4,6-disubstituted antibiotics; however, phosphorylation does not necessarily result in bacterial resistance. Hence, the APH(2″)-Ia domain of the bifunctional AAC(6′)-Ie/APH(2″)-Ia enzyme is a bona fide GTP-dependent kinase with a narrow substrate profile, including only 4,6-disubstituted aminoglycosides.     

The 1.7 ? X-Ray Crystal Structure of the Porcine Factor VIII C2 Domain and Binding Analysis to Anti-Human C2 Domain Antibodies and Phospholipid Surfaces

The factor VIII C2 domain is essential for binding to activated platelet surfaces as well as the cofactor activity of factor VIII in blood coagulation. Inhibitory antibodies against the C2 domain commonly develop following factor VIII replacement therapy for hemophilia A patients, or they may spontaneously arise in cases of acquired hemophilia. Porcine factor VIII is an effective therapeutic for hemophilia patients with inhibitor due to its low cross-reactivity; however, the molecular basis for this behavior is poorly understood. In this study, the X-ray crystal structure of the porcine factor VIII C2 domain was determined, and superposition of the human and porcine C2 domains demonstrates that most surface-exposed differences cluster on the face harboring the “non-classical” antibody epitopes. Furthermore, antibody-binding results illustrate that the “classical” 3E6 antibody can bind both the human and porcine C2 domains, although the inhibitory titer to human factor VIII is 41 Bethesda Units (BU)/mg IgG versus 0.8 BU/mg IgG to porcine factor VIII, while the non-classical G99 antibody does not bind to the porcine C2 domain nor inhibit porcine factor VIII activity. Further structural analysis of differences between the electrostatic surface potentials suggest that the C2 domain binds to the negatively charged phospholipid surfaces of activated platelets primarily through the 3E6 epitope region. In contrast, the G99 face, which contains residue 2227, should be distal to the membrane surface. Phospholipid binding assays indicate that both porcine and human factor VIII C2 domains bind with comparable affinities, and the human K2227A and K2227E mutants bind to phospholipid surfaces with similar affinities as well. Lastly, the G99 IgG bound to PS-immobilized factor VIII C2 domain with an apparent dissociation constant of 15.5 nM, whereas 3E6 antibody binding to PS-bound C2 domain was not observed.     

Synthesis of Certain Disaccharides Containing a α-or β- Rhamnopyranosidic Group and the Substrate Specificity of α-l-Rhamnosidase from Aspergillus niger

To investigate the substrate specificity of α-l-rhamnosidase from Aspergillus niger, the following seven substrates were synthesized: methyl 3-O-α-l-rhamnopyranosyl-α-d-mannopyranoside (1), methyl 3-O-α-l-rhamnopyranosyl-α-l-xylopyranoside (2), methyl 3-0-α-l-rhamnopyranosyl-α-l-rhamnopyranoside (3), methyl 4-0-α-l-rhamnopyranosyl-α-d-galactopyranoside (4), methyl 4-O-α-l-rhamnopyranosyl-α-d-mannopyranoside (5), methyl 4-0-α-l-rhamnopyra-nosyl-α-d-xylopyranoside (6), and 6-0-β-l-rhamnopyranosyl-d-mannopyranose (7). Compounds 1~6 were well-hydrolyzed by the crude enzyme, but 7 was unaffected.